Project Summary Clostridium difficile, a major nosocomial pathogen is the principal causative agent of antibiotic associated diarrhea and pseudomembranous colitis. The toxigenic C. difficile strains that cause disease secrete virulence factors, toxin A and toxin B, which are responsible for colonic injury and inflammation. C. difficile toxins have no export signature and are secreted by an unusual mechanism that involves TcdE, a holin-like protein. Like other channel forming proteins, TcdE exists as the oligomer in C. difficile membrane. The preliminary study found tcdE mRNA to contain multiple start-codons that translate into three different isoforms. It was also noted that tcdE over-expression in bacteria causes membrane damage. Based on these observations, it was hypothesized that TcdE content in C. difficile is highly controlled through various regulatory mechanisms. Aim 1 of this application will determine how transcription of a tcdE gene is regulated and will also study the regulatory mechanisms involved in the translation control of tcdE mRNA into different TcdE isoforms. Aim 2 will determine the importance of TcdE in C. diffcile pathogenesis by employing the well standardized C. difficile hamster infection model. Since TcdE has a prominent role in toxin secretion, this study can help us to understand the toxin secretion mechanism and its significance in C. difficile pathogenesis.